Throughout this application various publications are referred to in parenthesis. Full citations for these references may be found at the end of the specification. The disclosures of these publications are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains.
Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB), is the leading cause of mortality due to bacterial pathogens, claiming about two million lives annually. With the advent of the antibiotic era, TB became treatable and at one point eradication was believed possible. However, in recent years TB has reemerged as a major global health threat due to the disastrous combination of poverty, a deadly synergy with HIV, and the emergence of extensively-drug-resistant strains (XDR-TB) that are virtually untreatable with current chemotherapies (1). To counter this resurgence of TB and to combat XDR-TB, new treatment options are urgently needed based on novel classes of bacterial targets very different from those of the antibiotics currently in use.
Identification of essential gene functions required for in vitro growth of Mtb is a pivotal strategy for discovering new drug targets due to the ease of screening for antibacterial compounds and testing for resistance. A genome-wide screen of a saturated Mtb transposon mutant library using microarray-based transposon site hybridization (TraSH) technology indicated that more than 600 genes (˜15%) may be required for in vitro growth (2). However, the rational development of specific inhibitors is hampered by ignorance of the functions of many of these essential candidates. Identification of nonessential gene functions that are dispensable for in vitro growth but are required for virulence is an additional valuable source of drug targets, but inhibitor screening for these candidates is typically much more difficult. Using the TraSH technique, 194 of the nonessential Mtb genes were found to be specifically required for in vivo growth in mice (3). Given that Mtb is an obligate intracellular pathogen highly adapted to the human host, with no known significant environmental niche, the large number of genes dispensable for in vitro growth and virulence is surprising. This high degree of gene dispensability probably reflects extensive genetic redundancy or functional homeostatic buffering within essential processes, so that mutations in single genes are often compensated for by other genes. This means that many “synthetic lethal” pathways are certainly present in Mtb. “Synthetic lethality” describes any combination of two separately nonlethal mutations that jointly lead to inviability. In yeast, striking synthetic lethal genetic interactions in genome-wide studies have been demonstrated, revealing that most nonessential genes have several synthetic lethal interactions with other genes (4, 5). Identification of synthetic lethal pathways in Mtb would thus greatly increase the repertoire of drug target candidates.
New chemotherapeutics are urgently required to control the tuberculosis pandemic fueled by the emergence of multidrug- and extensively-drug-resistant Mtb strains and the bacterium's catastrophic alliance with HIV. The present invention answers this need by providing new assays for the development of chemotherapeutics to combat tuberculosis and other opportunistic pathogens. The present invention provides assays for the development of chemotherapeutics targeting the ubiquitous and conserved GlgE and Rv3032 α-glucan pathways.